Fifty-three cDNA fragments previously obtained by hybridization selection from random clones in the chromosome 21 cosmid library LL21CNO2 failed to identify clones in the chromosome 21 YAC contig described by Chumakov et al. (1992, Nature 359: 380-387). Using an expanded panel of somatic cell hybrids, we have verified that the majority of these cDNAs map to chromosome 21 and that in particular a very high proportion, approximately 85%, localize to a 5-Mb region of distal 21q22.3. Pulsed-field analysis coupled with information from the NotI restriction map of the region further indicate that 17 cDNA fragments map within 650 kb of the PFKL gene and thus may be candidates for genetic diseases linked to this gene. This work helps to characterize a region poorly represented in the CEPH YAC contig and adds to the number of cDNAs useful in analysis of chromosome 21-associated diseases.